Medicament for the two-step perioperative therapy of solid tumours

ABSTRACT

The present invention describes the use of an agent endowed with tumour tropism in combination with another agent with anticancer activity and with an affinity for the first agent for the preparation of a medicament useful for the two-step perioperative therapy of solid tumours. The advantages of the present invention consist in the greater, effective localisation of its anticancer activity, in the reduction of the number of administration steps and in the possibility of reducing the anticancer doses, with a resulting decrease in side effects, but without any loss of efficacy.

The invention described herein relates to the use of reagents for thepreparation of a medicament useful for intra- and postoperativelocoregional and systemic therapy.

BACKGROUND TO THE INVENTION

Conservative surgery with axillary dissection and supplementaryradiotherapy is the treatment of choice in patients with small-sizedbreast cancers. The results of recent clinical trials, particularlyrandomised trials (U. Veronesi, et al., New Engl. J. Med., 305:6-11,1981; U. Veronesi, et al., Ann. Surg. Vol. 211, 3:250-259, 1990), haveshown that the risk of local recurrence of the tumour correlates withthe extent of the operation on the breast, with the patient's age, andwith the presence of an extensive intraductal component and peritumorallymphatic and/or vascular invasion. In addition, a reduced incidence oflocal recurrence has been demonstrated in subjects undergoingsupplementary radiotherapy (5.4% as against 21.6% in the control group).

The supplementary external radiotherapy currently used after surgicalquadrantectomy involves the administration of a total dose of 50-60 Gyin 6 weeks of treatment, with irradiation of the entire residual breastafter surgery and optionally an overdose on the operative bed. This kindof treatment regimen has by no means negligible psychologicalimplications; its long duration increases and prolongs the patient'sstate of anxiety related to her experience of the disease and leads herto believe that the surgical operation has not been successful inresolving the disease. Moreover, there is also a by no means negligiblesocial impact in terms of costs, related to the patient's absence offwork for a period of about 2 months.

As an alternative to traditional radiotherapy treatment IntraoperativeRadiotherapy (IORT) has recently been proposed, which is a radiotherapytechnique that makes it possible to deliver a single dose of radiationdirectly to the tumour exposed during surgery, or to the anatomical areathat contained the tumour after surgical removal of the cancer. Theinventors of the present invention have used IORT with satisfactoryresults in the context of a randomised study for the treatment of stageT1 cancer of the breast. From the logistic point of view, however, onlyin a very few centres is it possible to implement this type of therapy;the cost of the equipment alone is more than one million euros, withoutconsidering the architectural costs of constructing a shielded operatingtheatre to guarantee radioprotection for the operators and people in theadjacent rooms and of the specialist staff necessary for implementingthe treatment.

A radioimmunotherapy protocol called three-step radioimmunotherapy isknown, the details of which are described in the European Journal ofNuclear Medicament Vol. 26, No. 2, February 1999, pp. 110-120 e No. 4,April 1999, pp. 348-357 and in European Patent EP 0 496 074. In thismethod, a reagent kit is used in a form suitable for intravenousadministration, consisting of 1) a biotinylated monoclonal antibodyspecific for an antigen associated with a tumour, 2) a protein of theavidin type, 3) biotin or one of its derivatives conjugated with anefficacious agent for the treatment and/or diagnosis of a tumour. Usefulagents for reducing the circulating levels of biotinylated antibody andof proteins of the avidin family (chasing agents) have also beendescribed in the three-step radioimmunotherapy method. Such kits areindicated for intracavitary or systemic administration, but nosuggestions are provided with regard to sequential administrationincluding both the surgical act and postoperative systemic treatment.

The three-step method is undoubtedly valid in its general description,but it can be optimised and further exploited if large amounts of avidinare introduced onto the tumour or in areas of the body which couldreceive or which already harbour residual tumour cells after anapparently radical operation. The two main limitations of the classicthree-step method consist in the fact that only modest amounts ofantibodies and proteins of the avidin family,—most frequentlystreptavidin (steps 1 and 2)—reach the target after intravenousinoculation. The locoregional inoculation is applicable in naturalanatomical cavities such as the peritoneum, pleura, or bladder, or inpostoperative virtual cavities as in the case of brain tumours.

SUMMARY OF THE INVENTION

It has now been found that a perioperative-type approach, including anintraoperative locoregional step and a second postoperative systemicstep, is particularly advantageous in controlling local recurrences,and, surprisingly, it has been seen that local therapy can beadvantageously administered in only two steps, the first of which isperformed intraoperatively in a locoregional site and the secondpostoperatively via a systemic route.

The introduction, during surgery, of an agent endowed with tumourtropism, that is to say, capable of concentrating locally on the tumourcell or in the vicinity of it, immediately prepares, in the residualtissue around the tumour, a sort of receptor of our choosing ready toreceive, locally and in an extremely high concentration, the subsequentdose of actual anticancer agent administered intravenously. Theanticancer agent must be suitably directed to the site of the tumour,exploiting the affinity of the carrier agent for the receptorartificially created.

Therefore, one initial object of the present invention is the use of afirst agent endowed with tumour tropism in combination with a secondanticancer agent endowed with affinity for said first agent as activeingredients for the preparation of a medicament useful for two-stepperioperative therapy, the first step of which consists in anintraoperative locoregional step and the second in a postoperativesystemic step. In one preferred embodiment of the invention, the therapywill take the form of radiotherapy.

Another object of the present invention is a pharmaceutical compositioncontaining said active ingredients in separate containers (kits)suitable for sequential locoregional and systemic administration, saidcomposition constituting a medicament useful for the adjuvantperioperative therapy of operable or not completely removable solidtumours, such as, for instance, cancers of the breast, pancreas, lung,pleura, peritoneum, face and neck, bladder, brain and others.

Advantageously, the present invention solves the problems existing inthe present state of the art outlined above. An additional advantageinherent in the increased accumulation of the agent endowed with tumourtropism in the tumour site may consist in the possible reduction of theamount of anticancer agent used.

DETAILED DESCRIPTION OF THE INVENTION

In a first preferred embodiment of the invention, the therapy isimmunotherapy and particularly radioimmunotherapy. Within the context ofthis first embodiment, the agent endowed with tumour tropism is abiotinylated antibody specific for antigens associated with a tumour.

According to the present invention, in the intraoperative step, thebiotinylated antibody is administered, followed by avidin, thusconstructing the “artificial receptor” for the subsequent actualanticancer agent. In this case, the anticancer agent will be carried bybiotin, which will be contained in a chemical compound suitable forforming a complex with the anticancer agent and hereinafter referred toas biotin complex, and which will be administered systemically in thepostoperative step.

Biotin, in fact, concentrates locally only where avidin is present andin this case one can be certain that avidin is present in the area oneintends to treat, in that it was introduced by the surgeon a few hoursearlier (e.g. from 4 to 72 hours) during the operation. This is afurther advantage of the present invention as compared to therapy ingeneral, in that it drastically reduces the time elapsing betweenremoval of the primary tumour and subsequent adjuvant therapy.

The biotinylated antibody is preferably a monoclonal antibody. Theantibody may be murine, human or, optionally, chimeric. Specificantibodies against antigens associated with tumours are known andavailable on the market or can be prepared with methods known to expertsin the field, such as, for example, those described in Trikha M., etal., Monoclonal antibodies as therapeutics in oncology, Current OpinionBiotechnology 2002, 13:609-614. Examples of antibodies are provided inthe above-mentioned European Patent EP 0 696 074. In a first preferredembodiment, the antibody is antitenascin monoclonal antibody. Chimericor recombinant antibodies can also be used (Trikha M., et al., ibid.).

With a view to achieving maximum biotinylated antibody accumulationcapacity in the target area a mixture of biotinylated monoclonalantibodies can be used, directed against different tumour antigens oragainst proteins of the extracellular matrix, such as tenascin. Theseproteins are particularly copious within the tumour and thus constitutean ideal target also after removal of the main tumour mass.

Experts in the field are familiar with antibody biotinylation.

The avidin compound used may be avidin itself which is a commerciallyavailable protein. In addition to naturally occurring avidin, otherproteins of the same type can be used, e.g. streptavidin, or polymericderivatives of avidin, streptavidin, or their derivatives withpolyethylene glycol (PEG-ylated avidins; Chinol M., Br. J. Cancer 1998;78:189-197).

The anticancer agent, which in this case will be carried by the biotincompound, may be selected from those available in the field or may beany other anticancer agent. Examples of anticancer agents arechemotherapeutic agents in general, toxins, cytokines, such as IL-2,interferon, TNF, lymphocyte cells and radionuclides, which constitute apreferred example in the present invention.

The radioactive biotin may be biotin itself labelled with a radioactiveisotope by means of a special linker.

The radioisotope will be selected in relation to the specificapplication. Examples of suitable radioisotopes are Fe-52, Mn-52m,Co-55, Cu-64, Ga-67, Ga-68, Tc-99m, In-111, I-123, I-125, I-131, P-32,Sc-47, Cu-67, Y-90, Pd-109, Ag-111, I-131, Pm-149, Re-186, Re-188,At-211, Pb-212, Bi-212, Lu-177. As will be clear from the followingdescription, beta-emitting radioisotopes such as Yttrium-90 orLuthetium-177 are preferred.

Though the present invention is applicable to the radioimmunotherapy oftumours in general, one preferred application is for breast, lung,pleural and peritoneal tumours.

The reagent doses will be determined by the expert in the field. Theywill, however, be calculated so as to deliver a dose which is sure to betumoricidal against its target.

Indicatively, the containers of the various reagents are generally in aform suitable for injectable administration and contain an adequatequantity of reagent. In a preferred embodiment form, the container ofthe antibody will take the form of a special syringe equipped with oneor more needles to facilitate infiltration of the tumour bed andsurrounding tissue. Conveniently, the container can also be in a formsuitable for administration of the antibody as a spray.

Again by way of an example, the avidin container will be suitable forcontaining an adequate amount of avidin, indicatively in a ratio of 5:1to the antibody (e.g. 100 mg vs. 20 mg of antibody), optionally dilutedin saline solution. In a particularly preferred form, the container willtake the form of a special syringe suitable for successiveadministrations of precise volumes, e.g. 20 ml in the first phase andone or more further aliquots (10 ml) in a second phase of the surgery,e.g. on the resection margins or residues of diseased tissue whichcannot be removed surgically because of infiltration of vital organs.Conveniently, the container may also be in a form suitable for theadministration of avidin as a spray.

Preferably, the various containers, already containing the doses of theindividual active ingredients, will be contained in a single pack (kit)bearing the instructions for the modes of administration.

The biotinylated antibody is administered intraoperatively, and after acertain amount of time, e.g. 10 minutes, different doses of avidin areadministered, e.g. a first dose in the tumour bed, and a second doseafter a possible reconstruction intervention.

Similarly, in a subsequent embodiment of the invention, avidinatedantibody or avidinated antibody derivatives can be administereddirectly. There is a method described in the patent application by NeorxEP 0 251 494, in which a number of antibody derivatives are usedsystemically. The present invention differs from that method in that thestep leading to the accumulation of avidin in the tumour site occurs inthe operative step via the locoregional route, whereas only theadministration of the biotinylated drug occurs in the postoperativestep. With this procedure a kind of “artificial receptor” is created,capable of taking up radioactive biotin.

From a minimum of 4 hours to 2-3 days postoperatively, the patient willbe accompanied to the nuclear medicament department to start thepostoperative therapeutic step with radiolabelled biotin administeredsystemically. As an alternative to radiolabelled biotin, the biotin canbe used as a vehicle for anticancer agents, such as, for example,chemotherapeutic agents or toxins or anticancer cells (e.g. thepatient's lymphocytes). (DOTA)-⁹⁰Y/¹⁷⁷⁷Lu biotin will always beadministered intravenously. The initial activity will be 50 mCi, for ⁹⁰Yand 80 mCi for ¹⁷⁷Lu. On the basis of previous experience withradioimmunotherapy, these activities are ⅓ less than the maxim activitythat can be administered per cycle. The therapeutic window, therefore,may range from 50 to 100 mCi for ⁹⁰Y and from 80 to 150 mCi for ¹⁷⁷Lu.Before administering the radioactive biotin, a “chaser” of biotinylatedalbumin can be administered, as described in the above-mentioned studiesby Paganelli et al. and in EP 0 496 074, particularly, a fixed dose of25 mg of biotinylated albumin, 10-15 minutes earlier.

Similarly, in another embodiment of the invention, avidin can bedirectly administered during the intraoperative locoregional phase,since it is endowed with a certain amount of tumour tropism andtherefore concentrates in the therapeutic target sites.

Following these simplified procedures, many tumours that do not presentspecific antigens could still be treated with the reagents avidin andbiotin. The sequence of the subsequent events then remains unchanged,through the time interval elapsing between the operation and thesystemic therapy, which must be reduced to 4-24 hours, because theaccumulation in the tumour site may require less time. The systemicadministration of radiolabelled biotin or carrier agent with anticanceractivity will take place as described above.

In another of its embodiments, the present invention also provides forthe use of the above-mentioned active ingredients for the preparation ofa diagnostic composition for pretherapeutic biodistribution (dosimetricdiagnostic phase), in which the second agent is radiolabelled. In thecase of the use of beta and gamma emitters (e.g. Lu 177) such asbiotinylated drugs, the dosimetric phase can be carried out at the sametime as the therapeutic phase.

REFERENCES

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1. Use of a first agent endowed with tumour tropism in combination witha second anticancer agent endowed with affinity for said first agent asactive ingredients for the preparation of a medicament useful for thetwo-step perioperative therapy of solid tumours.
 2. Use according toclaim 1, in which said first agent is administered during anintraoperative step via the locoregenional route and said second agentis administered during a postoperative step via the systemic route. 3.Use according to claim 1, in which said agent endowed with tumourtropism contains avidin.
 4. Use according to claim 1, in which saidfirst agent is avidin and said second agent is a biotin compound bearingan anticancer agent.
 5. Use according to claim 1, in which saidanticancer agent is selected from the group consisting of radioisotopes,chemotherapeutic agents, toxins and anticancer cells.
 6. Use accordingto claim 5, in which said radioisotope is selected from the groupconsisting of Fe-52, Mn-52m, Co-55, Cu-64, Ga-67, Ga-68, Tc-99m, In-111,I-123, I-125, I-131, P-32, Sc-47, Cu-67, Y-90, Pd-109, Ag-111 I-131,Pm-149, Re-186, Re-188, At-211, Pb-212, Bi-212, and Lu-177.
 7. Useaccording to claim 6, in which said radioisotope is Y-90 or Lu-177. 8.Use according to claim 4, in which the avidin and the biotin compound insaid medicament are in separate containers.
 9. Use according to claim 1,in which said tumour is selected from the group consisting of breast,pancreas, lung, pleural, peritoneal, cervico-facial, brain and bladdertumours.
 10. Use according to claim 3, in which said avidin is selectedfrom the group consisting of avidin, streptavidin, their polymericderivatives and their derivatives with polyethylene glycol.
 11. Useaccording to claim 1, in which said medicament is suitable forinjectable administration.
 12. Use according to claim 11, in which thecontainer of said avidin is in the form of a syringe suitable forsuccessive administrations of precise volumes.
 13. Use according toclaim 4, in which said avidin is contained in a separate container in asingle dose.
 14. Use according to claim 4, in which the container ofsaid avidin is suitable for administration in the form of a spray. 15.Kit for two-step perioperative therapy, consisting of a set of separatecontainers, in which a first container contains an avidin compound, anda second container contains a biotin compound bearing an anticanceragent.
 16. Kit according to claim 15, in which the first containercontains an avidinated antibody specific for antigens associated with atumour or a mixture of antibodies, and the second container contains abiotin compound bearing an anticancer agent.
 17. Use of a first agentendowed with tumour tropism in combination with a second radiolabelledagent endowed with affinity for said first agent for the preparation ofa diagnostic composition for the pretherapeutic biodistribution of themedicanient according to claim 1.